Figure 3

FF domains of Prp40 bind to specific peptides within Snu71 and Luc7. A, Peptides of Snu71-I (aa 329–536) and Luc7-II (aa 93–261) were synthesised as 15 mers with 3 aa shifts on a β-alanine membrane. A control peptide (QRALAKDLIVPRRP, position C17) binds to the α-GST antibody. Incubation with GST-FF1-2 showed a strong signal at peptide B1 and A24 (panel B). B, Peptide scan as in A but with Luc7 peptides probed with GST-FF1-2. Control membranes probed with GST alone were negative (data not shown). C, Alanine-scan of the peptide found in B (position A24, wildtype sequence: DRRLADHFLGKIHLG). Letters mark the amino acids which were replaced by alanine. GST-FF1 served as probe. The most important amino acid for the interaction are shown in bold. D, Substitution analysis of the Luc7 peptide sequence from C, probed with GST-FF1. Each residue within this sequence was substituted by 20 naturally occurring L-amino acids. All spots in circles represent the wild-type amino acids. All other spots are single substitution analogs, with rows defining the sequence position that is substituted and columns defining the amino acid that replaces the wild-type residue. E, The Luc7 motif bound by the FF1 domain of Prp40 as found in D. ϕ stands for hydrophobic amino acids, green indicates hydrophobic, yellow indicates polar, blue indicates basic and orange indicates acidic amino acids. White letters on black background represent the wildtype amino acids within the sequence.